MUS81 generates a subset of MLH1-MLH3-independent crossovers in mammalian meiosis

Two eukaryotic pathways for processing double-strand breaks (DSBs) as crossovers have been described, one dependent on the MutL homologs Mlh1 and Mlh3, and the other on the structure-specific endonuclease Mus81. Mammalian MUS81 has been implicated in maintenance of genomic stability in somatic cells; however, little is known about its role during meiosis. Mus81-deficient mice were originally reported as being viable and fertile, with normal meiotic progression; however, a more detailed examination of meiotic progression in Mus81-null animals and WT controls reveals significant meiotic defects in the mutants. These include smaller testis size, a depletion of mature epididymal sperm, significantly upregulated accumulation of MLH1 on chromosomes from pachytene meiocytes in an interference-independent fashion, and a subset of meiotic DSBs that fail to be repaired. Interestingly, chiasmata numbers in spermatocytes from Mus81-/- animals are normal, suggesting additional integrated mechanisms controlling the two distinct crossover pathways. This study is the first in-depth analysis of meiotic progression in Mus81-nullizygous mice, and our results implicate the MUS81 pathway as a regulator of crossover frequency and placement in mammals.     

Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein

During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.     

Proteolysis of the mismatch repair protein MLH1 by caspase-3 promotes DNA damage-induced apoptosis

Caspases are critical proapoptotic proteases that execute cell death signals by selectively cleaving proteins at Asp residues to alter their function. Caspases trigger apoptotic chromatin degradation by activating caspase-activated DNase and by inactivating a number of enzymes that sense or repair DNA damage. We have identified the mismatch repair protein MLH1 as a novel caspase-3 substrate by screening small pools of a human prostate adenocarcinoma cDNA library for cDNAs encoding caspase substrates. In this report, we demonstrate that human MLH1 is specifically cleaved by caspase-3 at Asp(418) in vitro. Furthermore, MLH1 is rapidly proteolyzed by caspase-3 in cancer cells induced to undergo apoptosis by treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the topoisomerase II inhibitor etoposide, which damages DNA. Importantly, proteolysis of MLH1 by caspase-3 triggers its partial redistribution from the nucleus to the cytoplasm and generates a proapoptotic carboxyl-terminal product. In addition, we demonstrate that a caspase-3 cleavage-resistant D418E MLH1 mutant inhibits etoposide-induced apoptosis but has little effect on TRAIL-induced apoptosis. These results indicate that the proteolysis of MLH1 by caspase-3 plays a functionally important and previously unrecognized role in the execution of DNA damage-induced apoptosis.     

Cytoskeletal scaffolding proteins interact with Lynch‐Syndrome associated mismatch repair protein MLH1

The involvement of MLH1 in several mismatch repair‐independent cellular processes has been reported. In an attempt to gain further insight into the protein's cellular functions, we screened for novel interacting partners of MLH1 utilizing a bacterial two‐hybrid system. Numerous unknown interacting proteins were identified, suggesting novel biological roles of MLH1. The network of MLH1 and its partner proteins involves a multitude of cellular processes. Integration of our data with the “General Repository for Interaction Datasets” highlighted that MLH1 exhibits relationships to three interacting pairs of proteins involved in cytoskeletal and filament organization: Thymosin β 4 and Actin γ, Cathepsin B and Annexin A2 as well as Spectrin α and Desmin. Coimmunoprecipitation and colocalization experiments validated the interaction of MLH1 with these proteins. Differential mRNA levels of many of the identified proteins, detected by microarray analysis comparing MLH1‐deficient and ‐proficient cell lines, support the assumed interplay of MLH1 and the identified candidate proteins. By siRNA knock down of MLH1, we demonstrated the functional impact of MLH1–Actin interaction on filament organization and propose that dysregulation of MLH1 plays an essential role in cytoskeleton dynamics. Our data suggest novel roles of MLH1 in cellular organization and colorectal cancerogenesis.     

The Bloom's syndrome protein (BLM) interacts with MLH1 but is not required for DNA mismatch repair

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by pre- and postnatal growth deficiency, immunodeficiency, and a tremendous predisposition to a wide variety of cancers. Cells from BS individuals are characterized by a high incidence of chromosomal gaps and breaks, elevated sister chromatid exchange, quadriradial formations, and locus-specific mutations. BS is the consequence of mutations that lead to loss of function of BLM, a gene encoding a helicase with homology to the RecQ helicase family. To delineate the role of BLM in DNA replication, recombination, and repair we used a yeast two-hybrid screen to identify potential protein partners of the BLM helicase. The C terminus of BLM interacts directly with MLH1 in the yeast-two hybrid assay; far Western analysis and co-immunoprecipitations confirmed the interaction. Cell extracts deficient in BLM were competent for DNA mismatch repair. These data suggest that the BLM helicase and MLH1 function together in replication, recombination, or DNA repair events independent of single base mismatch repair.     

Mutations in the MutSalpha interaction interface of MLH1 can abolish DNA mismatch repair

MutLα, a heterodimer of MLH1 and PMS2, plays a central role in human DNA mismatch repair. It interacts ATP-dependently with the mismatch detector MutSα and assembles and controls further repair enzymes. We tested if the interaction of MutLα with DNA-bound MutSα is impaired by cancer-associated mutations in MLH1, and identified one mutation (Ala128Pro) which abolished interaction as well as mismatch repair activity. Further examinations revealed three more residues whose mutation interfered with interaction. Homology modelling of MLH1 showed that all residues clustered in a small accessible surface patch, suggesting that the major interaction interface of MutLα for MutSα is located on the edge of an extensive β-sheet that backs the MLH1 ATP binding pocket. Bioinformatic analysis confirmed that this patch corresponds to a conserved potential protein–protein interaction interface which is present in both human MLH1 and its E.coli homologue MutL. MutL could be site-specifically crosslinked to MutS from this patch, confirming that the bacterial MutL–MutS complex is established by the corresponding interface in MutL. This is the first study that identifies the conserved major MutLα–MutSα interaction interface in MLH1 and demonstrates that mutations in this interface can affect interaction and mismatch repair, and thereby can also contribute to cancer development.     

Direct association of Bloom’s syndrome gene product with the human mismatch repair protein MLH1

Bloom’s syndrome (BS) is a rare genetic disorder characterised by genomic instability and cancer susceptibility. BLM, the gene mutated in BS, encodes a member of the RecQ family of DNA helicases. Here, we identify hMLH1, which is involved in mismatch repair (MMR) and recombination, as a protein that directly interacts with BLM both in vivo and in vitro, and that the two proteins co-localise to discrete nuclear foci. The interaction between BLM and hMLH1 appears to have been evolutionarily conserved, as Sgs1p, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Mlh1p. However, cell extracts derived from BS patients show no obvious defects in MMR compared to wild-type- and BLM-complemented BS cell extracts. We conclude that the hMLH1–BLM interaction is not essential for post-replicative MMR, but, more likely, is required for some aspect of genetic recombination.     

一组多功能的错配修复蛋白

错配修复蛋白是DNA错配修复系统中主要功能蛋白质,主要参与DNA复制过程中对错配碱基的识别和修复.近年来研究表明错配修复蛋白还参与DNA损伤信号的传递、细胞周期的调控、减数分裂和有丝分裂等.错配修复蛋白缺陷会增加患肿瘤的危险性或者直接导致肿瘤;由于错配修复蛋白参与了DNA损伤信号传递、周期调控,错配修复蛋白缺陷还会导致细胞对相关抗癌药物产生耐受.     

Rad9 plays an important role in DNA mismatch repair through physical interaction with MLH1

Rad9 is conserved from yeast to humans and plays roles in DNA repair (homologous recombination repair, and base-pair excision repair) and cell cycle checkpoint controls. It has not previously been reported whether Rad9 is involved in DNA mismatch repair (MMR). In this study, we have demonstrated that both human and mouse Rad9 interacts physically with the MMR protein MLH1. Disruption of the interaction by a single-point mutation in Rad9 leads to significantly reduced MMR activity. This disruption does not affect S/M checkpoint control and the first round of G₂/M checkpoint control, nor does it alter cell sensitivity to UV light, gamma rays or hydroxyurea. Our data indicate that Rad9 is an important factor in MMR and carries out its MMR function specifically through interaction with MLH1.     

Analysis of conditional mutations in the Saccharomyces cerevisiae MLH1 gene in mismatch repair and in meiotic crossing over

In mismatch repair (MMR), members of the MLH gene family have been proposed to act as key molecular matchmakers to coordinate mismatch recognition with downstream repair functions that result in mispair excision. Two members of this gene family, MLH1 and MLH3, have also been implicated in meiotic crossing over. These diverse roles suggest that a mutational analysis of MLH genes could provide reagents required to identify interactions between gene products and to test whether the different roles ascribed to a subset of these genes can be separated. In this report we show that in Saccharomyces cerevisiae the mlh1Delta mutation confers inviability in pol3-01 strain backgrounds that are defective in the Poldelta proofreading exonuclease activity. This phenotype was exploited to identify four mlh1 alleles that each confer a temperature-sensitive phenotype for viability in pol3-01 strains. In three different mutator assays, strains bearing conditional mlh1 alleles displayed wild-type or nearly wild-type mutation rates at 26 degrees. At 35 degrees, these strains exhibited mutation rates that approached those observed in mlh1Delta mutants. The mutator phenotype exhibited in mlh1-I296S strains was partially suppressed at 35 degrees by EXO1 overexpression. The mlh1-F228S and -I296S mutations conferred a separation-of-function phenotype in meiosis; both mlh1-F228S and -I296S strains displayed strong defects in meiotic mismatch repair but showed nearly wild-type levels of crossing over, suggesting that the conditional mutations differentially affected MLH1 functions. These genetic studies suggest that the conditional mlh1 mutations can be used to separate the MMR and meiotic crossing-over functions of MLH1 and to identify interactions between MLH1 and downstream repair components.     

Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae.

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.     

Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.     

Trans events associated with crossovers are revealed in the absence of mismatch repair genes in Saccharomyces cerevisiae

Genetic analysis of recombination in Saccharomyces cerevisiae has revealed products with structures not predicted by the double-strand break repair model of meiotic recombination. A particular type of recombinant containing trans heteroduplex DNA has been observed at two loci. Trans events were originally identified only in tetrads in which the non-Mendelian segregations were not associated with a crossover. Because of this, these events were proposed to have arisen from the unwinding of double Holliday junctions. Previous studies used palindromes, refractory to mismatch repair, as genetic markers whereas we have used a complementary approach of deleting mismatch repair proteins to identify heteroduplex DNA. We found that the markers occurred in trans and were associated with crossovers. In both mlh1Delta and msh2Delta strains, the frequency of trans events associated with a crossover exceeded that predicted from the random association of crossovers with noncrossover trans events. We propose two different models to account for trans events associated with crossovers and discuss the relevance to wild-type DSB repair.     

Evidence that a mutation in the MLH1 3'-untranslated region confers a mutator phenotype and mismatch repair deficiency in patients with relapsed leukemia

Defects in DNA mismatch repair (MMR) are the molecular basis of certain cancers, including hematological malignancies. The defects are often caused by mutations in coding regions of MMR genes or promoter methylation of the genes. However, in many cases, despite that a hypermutable phenotype is detected in a patient, no mutations/hypermethylations of MMR genes can be detected. We report here a novel mechanism that a mutation in the MLH1 3'-untranslated region (3'-UTR) leads to MMR deficiency. A relapsed leukemia patient displayed microsatellite instability, but no genetic and epigenetic alterations in key MMR genes were identifiable. Instead, a 3-nucleotide (TTC) deletion in the MLH1 3'-UTR was found in the patient's blood sample. The mutant MLH1 3'-UTR was found to significantly reduce the expressions of both a firefly luciferase reporter gene and an ectopic MLH1 gene in model cell lines. Consistent with these observations, a significant reduction in the steady-state level of MLH1 mRNA was observed in white blood cells of the patient. These findings suggest that the mutant MLH1 3'-UTR can cause a severely reduced/defective MMR activity conferring leukemia relapse, likely by down-regulating MLH1 expression at the mRNA level. Although the exact mechanism by which the mutant 3'-UTR down-regulates the MLH1 mRNA is not known, our findings provide a novel marker for cancers with MMR defects.     

Evidence for involvement of HMGB1 protein in human DNA mismatch repair

Defects in human DNA mismatch repair predispose to cancer, but many components of the pathway have not been identified. We report here the identification and characterization of a novel component required for mismatch repair in human cells. A 30-kDa protein was purified to homogeneity by virtue of its ability to complement a depleted HeLa extract in repair of mismatched heteroduplexes. The complementing activity was identified as HMGB1 (the high mobility group box 1 protein), a non-histone chromatin protein that facilitates protein-protein interactions and recognizes DNA damage. Evidence is also presented that HMGB1 physically interacts with MutSalpha and is required at a step prior to the excision of mispaired nucleotide in mismatch repair.     

Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene.

We have identified a new Saccharomyces cerevisiae gene, MLH1 (mutL homolog), that encodes a predicted protein product with sequence similarity to DNA mismatch repair proteins of bacteria (MutL and HexB) and S. cerevisiae yeast (PMS1). Disruption of the MLH1 gene results in elevated spontaneous mutation rates during vegetative growth as measured by forward mutation to canavanine resistance and reversion of the hom3-10 allele. Additionally, the mlh1 delta mutant displays a dramatic increase in the instability of simple sequence repeats, i.e., (GT)n (M. Strand, T. A. Prolla, R. M. Liskay, and T. D. Petes, Nature [London] 365:274-276, 1993). Meiotic studies indicate that disruption of the MLH1 gene in diploid strains causes increased spore lethality, presumably due to the accumulation of recessive lethal mutations, and increased postmeiotic segregation at each of four loci, the latter being indicative of inefficient repair of heteroduplex DNA generated during genetic recombination. mlh1 delta mutants, which should represent the null phenotype, show the same mutator and meiotic phenotypes as isogenic pms1 delta mutants. Interestingly, mutator and meiotic phenotypes of the mlh1 delta pms1 delta double mutant are indistinguishable from those of the mlh1 delta and pms1 delta single mutants. On the basis of our data, we suggest that in contrast to Escherichia coli, there are two MutL/HexB-like proteins in S. cerevisiae and that each is a required component of the same DNA mismatch repair pathway.     

Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain

Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.     

Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair

The human DNA mismatch repair (MMR) protein MLH1 has essential roles in the correction of replication errors and the activation of cell cycle checkpoints and cytotoxic responses to DNA damage that contribute to suppression of cancer risk. MLH1 functions as a heterodimer with the PMS2 protein, and steady state levels of PMS2 are very low in MLH1-deficient cells. Unique to MLH1 among MutL-homolog proteins, and conserved in identified eukaryotic MLH1 proteins, is the so-called C-terminal homology domain (CTH). The function of these C-terminal 20-30 amino acids is not known. We investigated the effect of a C-terminal truncation of human MLH1 (MLH1-L749X) on mammalian MMR by testing its activity in MLH1-deficient cells. We found the CTH to be essential for suppression of spontaneous mutation, activation of a cytotoxic response to 6-thioguanine, and maintenance of normal steady state levels of PMS2. Co-expression in doubly mutant Mlh1-/-; Pms2-/- fibroblasts showed that MLH1-L749X was unable to stabilize PMS2. Over-expression of MLH1-L749X did not reduce stabilization of PMS2 mediated by wild-type MLH1, indicating that truncation of the CTH reduces the ability to compete with wild-type MLH1 for interaction with PMS2. Lack of PMS2 stabilization also was observed with a previously reported pathogenic truncation (MLH1-Y750X), but not with two different point mutations in the CTH. Biochemical assays demonstrated that truncation of the CTH reduced the stability of heterodimers, although MLH1-L749X retained significant capacity for interaction with PMS2. Thus, the CTH of human MLH1 is necessary for error correction, checkpoint signaling, and for promoting interaction with, and the stability of, PMS2. Analysis of the CTH role in stabilizing PMS2 was facilitated by a novel intracellular assay for MLH1-PMS2 interaction. This assay should prove useful for identifying additional amino acids in MLH1 and PMS2 necessary for interaction in cells, and for determining the functional consequences of MLH1 mutations identified in human cancers.